cruzi VD lethal strain, either purified from mouse blood or from Vero cell cultures, 24 h-supernatants of blood and cellular trypomastigotes, and the VSV-G pseudotyped HIV-1 reporter vir
Trang 1Open Access
Research
Trypanosoma cruzi (Chagas' disease agent) reduces HIV-1
replication in human placenta
Guillermina Laura Dolcini*1, María Elisa Solana2, Guadalupe Andreani1,
Ana María Celentano2, Laura María Parodi3, Ana María Donato4,
Natalia Elissondo4, Stella Maris González Cappa2, Luis David Giavedoni3
and Liliana Martínez Peralta1
Address: 1 National Reference Center for AIDS, Microbiology Department, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina,
2 Laboratory of Parasitology, Microbiology Department, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina, 3 Department of Virology and Immunology, Southwest National Primate Research Center (SNPRC), Southwest Foundation for Biomedical Research (SFBR), San Antonio, Texas, USA and 4 Endocrinology Service, Department of Clinical Biochemistry, José de San Martín Hospital, School of Pharmacy and
Biochemistry, University of Buenos Aires, Buenos Aires, Argentina
Email: Guillermina Laura Dolcini* - gdolcini@fmed.uba.ar; María Elisa Solana - melisolana@yahoo.com.ar;
Guadalupe Andreani - gandreani@fmed.uba.ar; Ana María Celentano - amcele@fmed.uba.ar; Laura María Parodi - lparodi@sfbr.org;
Ana María Donato - donatoam@hotmail.com; Natalia Elissondo - natieli@hotmail.com; Stella Maris
González Cappa - smgcappa@fmed.uba.ar; Luis David Giavedoni - lgiavedo@sfbr.org; Liliana Martínez Peralta - lilimp@fmed.uba.ar
* Corresponding author
Abstract
Background: Several factors determine the risk of HIV mother-to-child transmission (MTCT),
such as coinfections in placentas from HIV-1 positive mothers with other pathogens Chagas'
disease is one of the most endemic zoonoses in Latin America, caused by the protozoan
Trypanosoma cruzi The purpose of the study was to determine whether T cruzi modifies HIV
infection of the placenta at the tissue or cellular level
Results: Simple and double infections were carried out on a placental histoculture system
(chorionic villi isolated from term placentas from HIV and Chagas negative mothers) and on the
choriocarcinoma BeWo cell line Trypomastigotes of T cruzi (VD lethal strain), either purified from
mouse blood or from Vero cell cultures, 24 h-supernatants of blood and cellular trypomastigotes,
and the VSV-G pseudotyped HIV-1 reporter virus were used for the coinfections Viral
transduction was evaluated by quantification of luciferase activity Coinfection with whole
trypomastigotes, either from mouse blood or from cell cultures, decreased viral pseudotype
luciferase activity in placental histocultures Similar results were obtained from BeWo cells
Supernatants of stimulated histocultures were used for the simultaneous determination of 29
cytokines and chemokines with the Luminex technology In histocultures infected with
trypomastigotes, as well as in coinfected tissues, IL-6, IL-8, IP-10 and MCP-1 production was
significantly lower than in controls or HIV-1 transducted tissue A similar decrease was observed
in histocultures treated with 24 h-supernatants of blood trypomastigotes, but not in coinfected
tissues
Conclusion: Our results demonstrated that the presence of an intracellular pathogen, such as T.
cruzi, is able to impair HIV-1 transduction in an in vitro system of human placental histoculture.
Published: 1 July 2008
Retrovirology 2008, 5:53 doi:10.1186/1742-4690-5-53
Received: 7 February 2008 Accepted: 1 July 2008 This article is available from: http://www.retrovirology.com/content/5/1/53
© 2008 Dolcini et al; licensee BioMed Central Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Trang 2Direct effects of the parasite on cellular structures as well as on cellular/viral proteins essential for
HIV-1 replication might influence viral transduction in this model Nonetheless, additional
mechanisms including modulation of cytokines/chemokines at placental level could not be excluded
in the inhibition observed Further experiments need to be conducted in order to elucidate the
mechanism(s) involved in this phenomenon Therefore, coinfection with T cruzi may have a
deleterious effect on HIV-1 transduction and thus could play an important role in viral outcome at
the placental level
Background
Mother-to-child transmission (MTCT) of human
immun-odeficiency virus type 1 (HIV-1) occurs mainly when the
newborn comes in contact with infected secretions of the
mother during birth, though HIV-1 can also be
transmit-ted through breastfeeding and in utero [1] MTCT rates
between 1–2% have been achieved after successful
appli-cation of preventive therapies, mainly in industrialized
countries [2-5] However, studies performed in large
cohorts with a follow-up of 8 years have shown that in
utero transmission may still occur before therapy is
initi-ated or effective [6] Thus, this type of transmission seems
to be a relevant way of MTCT even when efficient
antiret-roviral treatment and avoiding breastfeeding are being
successfully performed
The exact mechanisms by which the fetus acquires HIV-1
during pregnancy are not yet clear, even though the
pla-centa is an efficient natural barrier that plays a role in the
regulation of MTCT [7,8] Soluble factors in the placental
environment are part of this barrier Indeed, several
stud-ies have suggested that cytokines and chemokines may be
major regulators of transplacental transmission of HIV-1
[9-12] A recent study demonstrated that placental
explants from HIV-1 positive treated women secreted
higher levels of leukemia inhibitory factor (LIF),
inter-leukin (IL)-16, and regulated upon activation of normal T
cells expressed and secreted (RANTES), soluble factors
that inhibit HIV replication, and lower levels of TNF-α
and IL-8, proinflammatory factors known as stimulators
of viral replication [13]
Maternal viral load and immunological status are the
main factors that determine the risk of HIV-MTCT [14,15]
Other risk factors are coinfections of the mother [16,17],
an important issue since world regions with the highest
prevalence of HIV-1 infection are also affected by other
infections Thus, HIV positive pregnant women are
usu-ally infected with other pathogens, and such placental
coinfections may have consequences on MTCT of the
pathogens This is the case for HIV-1 infected pregnant
women of sub-Saharan Africa coinfected with Plasmodium
falciparum, who showed an increased peripheral and/or
placental viral replication with more adverse birth
out-comes than HIV uninfected women, particularly
multi-gravida women [18] Also noted, a shift in cytokine production towards a proinflammatory profile has been
associated with P falciparum placental infection [19,20],
which could stimulate HIV-1 replication [21]
In Latin America, one of the most important endemic pro-tozoonoses is Chagas' disease, caused by the protozoan
parasite Trypanosoma cruzi It extends from southern USA
to southern South America There are approximately 16–
18 million infected people, representing the largest para-sitic disease burden on the continent, with around 50,000 deaths per year and 100 million at risk of infection [22,23] Largely considered as a rural entity, Chagas' dis-ease has become an urban public health problem due to mass migration of rural inhabitants to big cities and an increase in poverty [24] This "urbanization" of Chagas' disease facilitates coinfection in the most important areas for HIV prevalence: the City of Buenos Aires and
sur-rounding areas T cruzi is mainly transmitted to humans
by vectors such as blood-sucking bugs present in rural areas, but also by blood transfusion or congenital trans-mission Due to the development of national programs for vector control and for the selection of blood donors, congenital transmission in women of child-bearing age
still remains a pressing public health issue since T cruzi
could be transmitted to their newborn throughout the course of infection [23] The rates of congenital transmis-sion vary from 1% to 10%, according to geographic areas [25] Such transmission takes place more frequently in the chronic stage of Chagas' disease, in endemic as well as in non-endemic areas, though its mechanisms have not been
clearly defined [24,26] In the case of T cruzi infected
mothers, no preventive treatment is possible during preg-nancy due to the antiparasitic drugs' toxicity for the fetus [27] Indeed, clinical management of these women differs greatly for HIV infected mothers
Data from HIV-T cruzi coinfected patients indicated
reac-tivation of parasite infection with exacerbation of clinical signs and unusual clinical manifestations [28-31] Even if
no evidence exist focus on clinical features of coinfected mothers, MTCT of both pathogens with severe outcome
for the children [32] and congenital transmission of T.
cruzi without confirmation of HIV-1 MTCT [33] were
reported However, little is known about interaction of
Trang 3both pathogens on an in vitro cellular or ex vivo tissue
model Thus, the purpose of the study was to determine
whether coinfection with T cruzi and HIV-1 at the tissue
or cellular level modifies HIV-1 infection
Results
Tissue viability and responsiveness to stimuli
Viability of the placental histocultures throughout the
cul-ture period was evaluated by quantifying total hCG
pro-duction in histoculture supernatants every 3 to 4 days
from day 1 to day 18 or 21 The maximum level of total
hCG was observed at day 4 or 7 A decrease was observed
between day 7 and day 11 of culture, and the minimum
level was reached after day 18 These levels are
compara-ble to those obtained in histocultures from previously
reported term placentas [34] Kinetics of hCG production
are shown in Figure 1
After the set-up of the histoculture system and before the
start of the infection protocols, the tissue response to an
external stimulus such as LPS was evaluated Placental
histocultures were stimulated with 0.1 and 10 μg/ml LPS
at day 0, 3 and 6 for 24 h before supernatant collection
Placental histocultures showed a response to this stimulus
in a dose-dependent manner secreting high amounts of
TNF-α at all the times tested (data not shown)
Tissue function is not modified by pseudotyped virus transduction and or parasite infection
After viral transduction with or without parasite infection, tissue function of placental histocultures was analyzed by measuring total hCG secretion levels in histoculture supernatants from each experiment As shown in Figure 2, neither viral nor parasite treatment significantly modified hCG secretion, indicating that the outcome of infection was not due to direct cytotoxicity of the inocula for the histocultures
T cruzi trypomastigotes and 24 h-supernatants of
trypomastigotes decrease HIV-1 replication in BeWo cells
The effect of blood trypomastigotes (BT) on HIV-1 repli-cation was assessed on BeWo cells, a model of early tro-phoblast cells which are the first placental layer in direct contact with maternal blood Previous data indicated that the HIV-1 R5 (BaL) or X4 (HXB2) pseudotyped reporter virus did not replicate in BeWo cells [35], thus we used only VSV-G pseudotyped HIV-1 reporter virus Cells were incubated with BT and/or pseudotyped virus, and trans-duction of the luciferase reporter gene as an indicator of viral replication was evaluated at the end of the experi-ment As shown in Figure 3 (left bar), viral replication was
decreased by BT (-86%, p < 0.005).
As trypomastigotes shed several soluble factors [36], we wanted to determine whether the trypomastigote super-natant could interfere with HIV-1 replicative cycle, or if an
active T cruzi infection was necessary to achieve the
previ-Production of hCG in the culture medium of placental histocultures
Figure 1
Production of hCG in the culture medium of placental histocultures Chorionic villi were placed on 1.5 cm2 collagen sponge gels at medium-air interface into the wells of 6-well plates, 9 blocks per collagen sponge and per well Production of hCG was measured in histoculture supernatants every 3 to 4 days from day 1 to day 18 or 21 by the chemiluminescence method Placental histocultures were maintained in 5% CO2 atmosphere/95% air at 37°C Results represent mean ± SD of duplicates and are representative of 3 independent experiments
Trang 4ously described effect Thus, BeWo cells were incubated
with 24 h-supernatants from BT (BTSn) and VSV-G
pseu-dotype virus Similar effect on luciferase activity as in the
case of BT was observed for BTSn (-76%, p < 0.005)
(Fig-ure 3, right bar)
T cruzi trypomastigotes and 24 h-supernatants of
trypomastigotes decrease HIV-1 replication in placental
histocultures
Transduction with pseudotyped virus harboring VSV-G,
HIV-1 R5 (BaL) or HIV-1 X4 (HXB2) envelope protein
were performed on placental histocultures, with or
with-out infections with BT Results were normalized in each
sample by total protein concentration When placental
histocultures were transducted with HXB2 pseudotyped
virus, no luciferase activity was detected (data not shown)
When BaL pseudotyped virus was used, even at higher
doses than VSV-G pseudotyped virus, levels of luciferase
activity were lower However, for both pseudotyped virus
luciferase activities were significantly decreased in
coinfec-tion with BT (mean ± SD; -90.98% ± 5.83, p < 0.001 for
VSV-G and -94% ± 5.02, p < 0.001 for BaL) (Figure 4A).
Purification of BT might carry other components from
mouse blood, mainly white cells and platelets, which
could interfere with HIV-1 replication Thus, similar
experiments were performed using trypomastigotes
puri-fied from Vero cell culture supernatants (CT) Similarly,
live CT significantly decreased virus-driven luciferase
Tissue functionality after pseudotyped virus transduction and/or parasite infection
Figure 2
Tissue functionality after pseudotyped virus transduction and/or parasite infection Placental villi were dissected
and immediately transducted overnight with VSV-G pseudotyped HIV-1 (V) (100 ng p24/placental block) alone or in the pres-ence of blood trypomastigotes (BT) (106 parasites/placental block) or 24 h-supernatant of BT (BTSn) After infection or coin-fection, tissue functionality of placental histocultures was analyzed by measuring total hCG secretion levels in histoculture supernatants by the chemiluminescence method at day 4 post-infection or coinfection Results represent mean ± SD of dupli-cates and are representative of 5 independent experiments
Effect of blood T cruzi trypomastigotes and 24 h-supernatant
of trypomastigotes on HIV-1 replication in BeWo cells
Figure 3
Effect of blood T cruzi trypomastigotes and 24
h-supernatant of trypomastigotes on HIV-1 replication
in BeWo cells The human choriocarcinoma BeWo cell line
was transducted overnight with VSV-G pseudotyped HIV-1 (V) (100 ng p24/2 × 104 cells per well) alone or in the pres-ence of blood trypomastigotes (BT) (2 × 105 parasites/2 ×
104cells per well) or 24 h-supernatant of BT (BTSn) Cells were lysed and luciferase activity as an indicator of viral repli-cation was read from cell lysates at day 4 post-infection or coinfection Results are expressed as relative light units per second (RLU/sec), presented as a percentage relative to
VSV-G The histogram in red corresponds to the % of infection with VSV-G (= 100%) and the histogram in white corre-sponds to the % of infection in the presence of BT Results are representative of 3 independent experiments
Trang 5Effect of blood and culture T cruzi trypomastigotes and 24 h-supernatants of trypomastigotes on HIV-1 replication in placental
histocultures
Figure 4
Effect of blood and culture T cruzi trypomastigotes and 24 h-supernatants of trypomastigotes on HIV-1
repli-cation in placental histocultures A: Placental villi were transducted overnight with BaL (B) (250 ng p24/placental block) or
VSV-G pseudotyped HIV-1 (V) (100 ng p24/placental block) alone or in the presence of blood trypomastigotes (BT) (106 para-sites/placental block) Fragments were homogenized and luciferase activity as an indicator of viral replication was read from tis-sue lysate at day 4 post-infection or coinfection Results are expressed as relative light units per second (RLU/sec), presented
as a percentage relative to B or V and were normalized in each sample by total protein concentration (RLU/prot) The histo-gram in red corresponds to the % of infection with BaL or VSV-G (= 100%) and the histohisto-gram in white corresponds to the %
of infection in the presence of BT B: Placental villi were transducted overnight with VSV-G pseudotyped HIV-1 (V) (100 ng
p24/placental block) alone or in the presence of blood trypomastigotes (BT) or cell trypomastigotes (CT) (106 parasites/placen-tal block), or 24 h-supernatants of BT (BTSn) or 24 h-supernatants of CT (CTSn) Fragments were homogenized and luciferase activity as an indicator of viral replication was read from tissue lysate at day 4 post-infection or coinfection Results are expressed as relative light units per second (RLU/sec), presented as a percentage relative to V and were normalized in each sample by total protein concentration (RLU/prot) The histogram in red corresponds to the % of infection with VSV-G (=
100%) and the histogram in white corresponds to the % of infection in the presence of BT, CT, BTSn or CTSn (p < 0.001)
Results are represented as a mean of 5 independent experiments
Trang 6activity in placental histocultures when they were
coin-fected with VSV-G pseudotyped HIV-1 reporter virus
(mean ± SD; -97.36% ± 0.98, p < 0.001) (Figure 4B).
Additionally, placental explants were incubated with 24
h-supernatants from either BT (BTSn) or CT (CTSn) and
VSV-G pseudotype virus A similar effect on luciferase
activity as in the case of BT was observed for BTSn (mean
of diminution ± SD; -81.48% ± 8.15, p < 0.001), while
CTSn also decreased luciferase activity although at a lower
degree (-61.21% ± 1.94, p < 0.001) (Figure 4B).
Effect of coinfection on soluble factor secretion
In an attempt to determine whether changes in the
pla-cental microenvironment due to parasite-viral interaction
are responsible for inhibiting HIV replication, cytokine/
chemokine secretion was measured in histoculture
super-natants at day 1 and at day 4 post-infection or coinfection
Results for day 1 presented in Figure 5 demonstrate that T.
cruzi acts as a potent inhibitor of IL-6 (p < 0.01), IL-8 (p <
0.05), IP10 (p < 0.01) and MCP-1 (p < 0.02), while no
sig-nificant changes were observed in RANTES, 1α, MIP-1β, G-CSF, GRO-α, GM-CSF, IFN-γ, 10, 13, MIP-1β,
IL-2, IL-4 and IL-5 production (data not shown) The effect
of HIV-T cruzi interaction on cytokine/chemokine
secre-tion seems to be parasite-driven since their levels corre-lated with those induced by the parasite alone
When placental histocultures were treated with BTSn,
sig-nificant decreases only in IL-6 (p < 0.04), IL-8 (p < 0.05), MCP-1 (p < 0.01), GM-CSF (p < 0.05), MIP-1α (p < 0.05), and MIP-1β (p < 0.05) secretion were detected in
histocul-ture supernatants collected at day 1 post-infection or coin-fection (Figure 6) Surprisingly, this diminution was detected only in BTSn treated histocultures but no changes were observed in HIV-BTSn treated samples
In all experiments, the differences seen at day 1 were no longer seen at day 4 post-infection or coinfection
Effect of blood T cruzi trypomastigotes on cytokine/chemokine secretion in placental histocultures
Figure 5
Effect of blood T cruzi trypomastigotes on cytokine/chemokine secretion in placental histocultures Placental villi
were transducted overnight with VSV-G pseudotyped HIV-1 (V) (100 ng p24/placental block) alone or in the presence of blood trypomastigotes (BT) (106 parasites/placental block) Histoculture supernatants were collected after infection or coinfection, diluted with 10% FCS in RPMI and used for the simultaneous determination of cytokine/chemokine production with Luminex
technology Results displayed correspond to IL-6 (p < 0.01), IL-8 (p < 0.05), IP10 (p < 0.01) and MCP-1 (p < 0.02) Results are
expressed as the mean ± SD and are representative of 5 independent experiments
Trang 7As a result of the significant burden of the HIV pandemics
in resource-poor regions, a number of potential
epidemi-ological, biepidemi-ological, and clinical interactions between HIV
and other tropical pathogens gained relevance and need
to be studied The interactions between HIV and tropical infectious agents are complex Each pathogen has the potential to alter the epidemiology, natural history, and/
or response to therapy of the other pathogens [37]; there-fore, it is unpredictable to establish the outcome of such
Effect of 24 h-supernatants of blood T cruzi trypomastigotes on cytokine/chemokine secretion in placental histocultures
Figure 6
Effect of 24 h-supernatants of blood T cruzi trypomastigotes on cytokine/chemokine secretion in placental
histocultures Placental villi were transducted overnight with VSV-G pseudotyped HIV-1 (V) (100 ng p24/placental block)
alone or in the presence of 24 h-supernatants of blood trypomastigotes (BTSn) (106 parasites/placental block) Histoculture supernatants were collected after infection or coinfection, diluted with 10% FCS in RPMI and used for the simultaneous
deter-mination of cytokine/chemokine production with Luminex technology Results displayed correspond to IL-6 (p < 0.04), IL-8 (p
< 0.05), MIP-1α (p < 0.05), MIP-1β (p < 0.05), GM-CSF (p < 0.05) and MCP-1 (p < 0.01) (pg/ml) Results are expressed as the
mean ± SD and are representative of 5 independent experiments
Trang 8coinfections In Latin America, one of the most significant
endemic protozoonoses is Chagas' disease, and several
clinical studies from HIV-T cruzi coinfected patients have
been reported [28-31] MTCT is one way of transmission
shared by both pathogens The exact mechanisms
involved in MTCT of both pathogens are not clear Hence,
the study of their interaction at the placental level is
criti-cal for designing strategies that abolish MTCT
In our in vitro culture system of term placental
histocul-tures, as well as in the trophoblast cell line BeWo, we
dem-onstrate that acute coinfection with T cruzi and HIV-1
pseudotyped virus decreases HIV-1 replication This is the
first report about interaction of these pathogens at the
pla-cental level
In order to validate our placenta in vitro model, we
evalu-ated the viability and responsiveness to stimuli of the
histocultures As previously described [34], micro-explant
villi of term placentas were morphologically viable until
day 7 or 11 with no significant alterations (data not
shown) and total hCG secretions peaked at day 7 or 11
Additionally, they were able to react to external stimuli
such as LPS, secreting a great amount of TNF-α, as
prously described [38] Altogether, these results provide
evi-dence that placental villi are intact and remain viable and
functional until at least day 7 of culture
For BeWo cells and placental histoculture infection, a
pseudotyped Vesicular Stomatitis Virus G/HIV-1 was
used This pseudotyped virus, due to its amphotropic
nature, is able to infect any cell type, regardless of receptor
and coreceptor surface expression [39] When R5-Env
pseudotyped HIV-1 was used on placental histocultures,
coinfection with trypomastigotes also abolished HIV-1
replication almost completely (Fig 4A) Previous studies
have demonstrated that X4-Env HIV-1 pseudotype viruses
do not infect human term placental chorionic villi and
that a higher dose of R5-Env HIV-1 pseudotypes,
com-pared to viruses pseudotyped with VSV-G, is necessary to
observe an infection of placental tissue [21] Similarly,
previous studies have shown that malignantly
trans-formed human cell lines of the trophoblast lineage are
resistant to cell-free HIV-1 pseudotypes bearing the R5
and X4 envelopes, and that this resistance was bypassed
when HIV-1 envelopes were substituted by the VSV-G
pro-tein [35] Thus, subsequent experiments of our study on
placental histocultures as well as on BeWo cell line were
performed only with VSV-G pseudotyped virus Although
we do not address the effects of T cruzi on the binding of
HIV-1 to their natural receptors, our experiments are valid
in studying the effects of the parasite on viral replication
A great impairment of HIV-1 replication was observed in
coinfection with viable T cruzi trypomastigotes purified
from mouse blood (BT) Moreover, when other source of viable trypomastigotes was used, such as those grown in cell culture (CT), the same effect on HIV-1 replication was observed In all cases, hCG secretion was measured in histoculture supernatants and no significant differences were observed between control, viral infection, or treat-ment with trypomastigotes These results indicate that pla-cental tissue remains viable and that parasite impairment
of HIV-1 replication was not associated with direct cell
toxicity caused by T cruzi Previous data indicate that the
parasite induces rearrangement of cortical cytoskeleton of syncytiotrophoblast with actin microfilament depletion during human placental invasion [40]
Considering that after entering the cell, the HIV-1 virion interacts initially with actin filaments which assist binding
to microtubules and transport to the nuclear periphery [41], modifications in trophoblast cytoskeleton might impair viral replication at an early phase in the case of
active T.cruzi invasion However, the inhibition of HIV
replication seems to be caused not only by viable trypo-mastigotes but also by soluble factors shed by the parasite, either from BT or CT
Taking into account that T cruzi sheds several proteases
(cysteine, serine, threonine and metalloproteinases) that participate in host cell invasion [36], those molecules could interfere with some critical structures inside the cytosol of host cells required for viral genome retrotran-scription and transfer to the nucleus Indeed, this is allowed by the reverse transcription complex that later becomes the preintegration complex, and both complexes include not only viral RNA or DNA and several accessory viral proteins, but also cellular proteins [42], which are necessary for efficient reverse transcription of HIV-1 [43] Disruption of these complexes by exogenous enzymes or alteration of protein interactions can lead to an impaired HIV-1 replication [44] We might hypothesize that this is
the case when T cruzi proteases are present.
Since the T cruzi is a complex intracellular organism that
has a great impact on host cell structure and also on its metabolism, we decided to evaluate whether the parasite
or its soluble products are able to modify the placental environment In fact, many soluble factors, including cytokines and hormones, with regulatory activities are essential for establishing and maintaining pregnancy [45,46] HIV-1 and antiretroviral treatment in pregnant women have an impact on the pattern of placental soluble factors [13,38] On the other hand, little is known about
the changes in human fetal-maternal interface in T cruzi
infection In histocultures infected with trypomastigotes and in coinfected tissue, IL-6, IL-8, IP-10 and MCP-1 pro-duction was significantly lower than in controls or HIV-1 infected tissues Certainly, most of these chemokines are
Trang 9an important driving-force for CD8+ T-cell recruitment,
which plays a significant role in the control of acute T.
cruzi infection [47] Concerning HIV-1, many reports
describe the role of cytokines/chemokines on its
replica-tion Among them, IL-6 is a well-known cytokine with
up-regulating activity on HIV replication [48] Moreover,
MCP-1 and IP-10 were associated with an increase in
leu-kocyte density and cerebrospinal fluid viral load [49-51]
Indeed, IP-10, as well as IL-8 may stimulate HIV-1
replica-tion in different cell types [52,53], although mechanisms
are not clearly defined In our system, a diminished
secre-tion of those stimulatory cytokines/chemokines was
observed at day 1 post-infection whenever the parasite
was present These transitory changes in the placental
environment might contribute to HIV-1 replication
impairment
Parallel studies have also been conducted on another
cel-lular system for the study of HIV/T cruzi interaction as
one of the main cell targets for both pathogens, the
mono-cyte-derived macrophages, and similar inhibition of viral
replication was observed at different levels of HIV-1
repli-cation cycle (Andreani G at al., manuscript in
prepara-tion) Thus, related mechanisms by which T cruzi impair
HIV-1 replication seem to be involved in different in vitro
systems
Conclusion
Our results demonstrate that the presence of an
intracellu-lar pathogen such as T cruzi is able to impair HIV-1
trans-duction in an in vitro system of human placental
histocultures Direct effects of the parasite on cellular
structures as well as on cellular/viral proteins essential for
HIV-1 replication might influence viral transduction in
this model Nonetheless, additional mechanisms
includ-ing modulation of cytokines/chemokines at placental
level could not be excluded in the inhibition observed
Further experiments need to be conducted in order to
clar-ify the mechanism(s) involved in this phenomenon
In summary, coinfection with T cruzi may have a
delete-rious effect on HIV-1 transduction and thus could play an
important role in viral outcome at the placental level
Methods
Histocultures of chorionic villi from term placentas
Term placentas were obtained after programmed cesarean
section at the Obstetrics Unit of the Fernández and Ramos
Mejía Hospitals in the city of Buenos Aires, in accordance
with Argentinean ethics guidelines This study was
approved by the Ethics Committee from the School of
Medicine, University of Buenos Aires Histocultures of
chorionic villi were performed as previously described
[34] with slight modifications Briefly, placental villi were
isolated, washed extensively with RPMI 1640 (CellGro, USA) and dissected into 2–3 mm blocks After infection or coinfection, chorionic villi were placed on 1.5 cm2 colla-gen sponge gels (Espongostan, Johnson & Johnson, USA)
at medium-air interface into the wells of 6- or 12-well plates (Greiner, Germany) with 3 or 2 ml media per well respectively, at 9 tissue blocks per collagen sponge and per well Histoculture medium was RPMI 1640 (CellGro) supplemented with 15% heat-inactivated fetal calf serum (FCS, PAA-Bioser, Argentina), 1% penicillin-streptomy-cin, 0.1% gentamipenicillin-streptomy-cin, 1% L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate; (Gibco BRL Ltd., USA) Placental histocultures were maintained in 5% CO2 atmosphere/95% air at 37°C Each experimental point means a duplicate histoculture well
Evaluation of histoculture viability and response to stimuli
Viability of histocultures was monitored by detection of total hCG secretion determined with a chemilumines-cence method (Immulite 1000, detection limit 1.1 mIU/
ml, Siemens Medical Solutions Diagnostics, USA) in supernatants from both histoculture system setup proto-cols (from day 1 to day 18) and in infection protoproto-cols (day 4 post-infection or coinfection) Tissue responses to LPS were analyzed by incubating tissue fragments with 0.1
and 10 μg/ml LPS (from E coli serotype 055:B5,
Sigma-Aldrich, USA) at day 0, 3 and 6 of histoculture After 24 h, levels of Tumor Necrosis Factor-alpha (TNF-α) secretion were quantified by ELISA (Peprotech, Mexico) in histocul-ture supernatants
Trophoblast cell line
The human choriocarcinoma BeWo cell line [54], used as
a model for early trophoblast cells [55-57], was obtained from the American Type Culture Collection (ATCC # CCL98, Rockville, Md.) These cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, CellGro) containing 25 mM glucose, supplemented with 20% heat-inactivated FCS, 20 mM glutamine, 50 IU/ml penicillin and 50 μg/ml streptomycin, in 5% CO2 atmosphere/95% air at 37°C
Pseudotyped viruses
Luciferase reporter viruses were produced as previously described [35] by transiently cotransfecting (SuperFect; Qiagen, Germany) 293T cells with the proviral
pNL-Luc-E-R+ vector [58], which lack the env gene and has the firefly luciferase gene inserted into the nef gene, and the
expres-sion vector pCMV harboring the gene coding for either the VSV-G envelope protein or the HIV-1 R5 (BaL) envelope protein [59], or the expression vector pSV harboring the gene coding for HIV-1 X4 (HXB2) envelope protein [60] Supernatants from 293T cells were harvested 72 h after
transfection and p24gag levels were measured using a
commercial ELISA kit (Murex, UK)
Trang 10T cruzi purification and supernatant preparation
T cruzi VD strain (isolated from a case of congenital
Cha-gas' disease, lethal for mice, lineage II) was used [61] This
subpopulation was maintained by serial passages in
21-day old CF1 mice Either bloodstream forms (BT) or tissue
culture-derived (CT) trypomastigotes were employed for
the coinfection assays BT were collected from blood of T.
cruzi infected mice at the peak of parasitemia by cardiac
puncture To enrich blood supernatants with BT, the
cen-trifuged blood was incubated for 1 h at 37°C and the
supernatant was collected Thus, BT were pelleted by
cen-trifugation for 30 min at 10,000 × g, counted in a
Neu-bauer hematocytometer and diluted to 107 BT/ml in
BeWo medium or to 4 × 107 BT/ml in histoculture
medium for further use in coinfection assays In order to
obtain CT, Vero cell monolayers were allowed to interact
with BT in a parasite/cell ratio of 5:1 for 24 h CT
har-vested from the second passage in Vero monolayers were
pelleted by centrifugation for 30 min at 10,000 × g,
counted in a Neubauer hematocytometer and diluted to 4
× 107 CT/ml in histoculture medium for further use in
coinfection assays
In order to obtain parasite supernatants, 107 BT diluted in
BeWo medium or 4 × 107 BT or CT diluted in histoculture
medium were incubated for 24 h at 37°C in 5% CO2 To
remove parasites and cellular debris, both parasite
sus-pensions were pelleted as described above and the
super-natants were filtered through a 0.22 μm pore-size filter
Filtrated aliquots were stored at -80°C until use for
coin-fection assays
Infection of trophoblast cells
BeWo cells were seeded in 96-well plates (2 × 104cells per
well) 24 h before infection and then incubated with
VSV-G pseudotyped HIV-1 (100 ng of p24 per well) and 2 ×
105 BT or 24 h-supernatant of BT (BTSn) overnight at
37°C in 5% CO2 Controls included transduction with
only the pseudotyped virus or Δenv pseudotype, infection
with parasites or treatment with parasite supernatants,
and also mock infected cells with culture medium After
culture for an additional 72 h, 100 μl of luciferase lysis
buffer (Promega) per well was added and luciferase
activ-ity as an indicator of viral replication was measured in 10
μl of lysate with a luminometer (Veritas), using the
com-mercially available substrate; data were expressed as RLU/
sec
Infection of placental histocultures
After dissection, 18 blocks of placental villi were placed in
24-well plates and transducted overnight with BaL (250
ng p24/placental block), HXB2 (250 ng p24/placental
block) or VSV-G pseudotyped HIV-1 (100 ng
p24/placen-tal block) and infected with BT or CT (106
parasites/pla-cental block), or parasite supernatants Controls included
transduction with only the pseudotyped virus or Δenv
pseudotype, infection with parasites or treatment with parasite supernatants, and also mock infected histocul-tures with culture medium The following day, placental blocks were washed 6 times in 6-well plates with PBS 1× (Gibco BRL Ltd.) using a cell strainer (BD Biosciences, USA), placed on collagen sponges and cultured as described above for an additional 72 h Protocols of over-night infection and then supernatant collection were also performed
For further cytokine/chemokines quantification, at the end of each experiment, supernatants were collected, clar-ified by centrifugation at 1,000 × g for 10 minutes, filtered (0,22 μm), aliquoted and stored at -80°C Placental frag-ments were collected and preserved at -80°C until homogenization
For luciferase activity quantification, placental fragments were homogenized in 500 μl of luciferase lysis buffer (Promega, USA) with an Ultra-turrax homogenizer (IKA, USA) Luciferase activity was measured at day 4 post-infec-tion or coinfecpost-infec-tion in 20 μl of lysate with a luminometer (Veritas, USA), using a commercially available substrate (Dual-Luciferase Reporter Assay System, Promega), and expressed as relative light units (RLU) Results were nor-malized to total protein concentration measured on lysates from each sample using a Micro BCA™ Protein Assay Kit (Pierce, USA) Final data were expressed as RLU/
μg prot (RLU/prot)
Quantification of the protein secretion of soluble factors
in placental histocultures
Supernatants of stimulated histocultures were diluted with 10% FCS in RPMI and used for the simultaneous determination of 29 cytokines and chemokines with Luminex technology as previously described [62,63] The coated bead/biotinylated antibody combinations used were: G-CSF (LINCOplex human G-CSF, Linco Research,
St Charles MO), GM-CSF (Beadlyte human GM-CSF, Upstate USA, Charlottesville, VA), GRO-α (Beadlyte human GRO-α, Upstate), IFN-α (anti-human IFN-α clones MMHA-11 and MMHA-2, PBL Biomedical Labora-tories, Piscataway, NJ), IFN-γ (Beadlyte primate IFN-γ, Upstate), IL-1β (Monkey IL-1β ELISPOT reagents, U-Cytech), IL-1Ra (Fluorokine MAP human IL-1Ra/IL-1F3, R&D System, Minneapolis, MN), IL-2 (Beadlyte primate IL-2, Upstate), IL-4 (LINCOplex human IL-4, Linco), IL-5 (LINCOplex human IL-5, Linco), IL-6 (LINCOplex human IL-6, Linco), IL-7 (anti-human IL-7 clone 7417 and polyclonal anti-human IL-7, R&D), IL-8 (Beadlyte human IL-8, Upstate), IL-9 (Beadlyte human IL-9, Upstate), IL-10 (anti-human IL-10 clones BN-10 and
QS-10, Cell Sciences Inc., Canton, MA), IL12(p40) (anti-human IL-12 clones IL-12I and IL-12II, Mabtech Inc.,